Abstract
.In the brain, the strength of each individual synapse is defined by the complement of proteins present or the “local proteome.” Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments.
Highlights
Neurons are highly specialized cells that communicate with one another at synapses, points of close contact between the axon of one neuron and the dendrite of another
It is known that the integrative properties of dendrites and synapses change as a function of distance from the neuronal cell body, but the proteomic landscape of synapses within a single cell is not known
Both limitations can be bypassed by DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT),[12] an extension of the original concept of PAINT13 that built on the repetitive and transient binding of a fluorophore to a target
Summary
Neurons are highly specialized cells that communicate with one another at synapses, points of close contact between the axon of one neuron and the dendrite of another. It is known that the integrative properties of dendrites and synapses change as a function of distance from the neuronal cell body, but the proteomic landscape of synapses within a single cell is not known. This gap is largely due to limitations in existing methods for quantifying and mapping proteins at the whole-cell level. Low numbers of singlemolecule events can affect the accuracy of molecular quantification,[9,10] and the resulting inaccuracy is exacerbated by the typical isolated fields of view.[11] Both limitations can be bypassed by DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT),[12] an extension of the original concept of PAINT13 that built on the repetitive and transient binding of a fluorophore to a target. As a proof of concept, we determine copy numbers of GluA2, an integral component of the (AMPA-type) glutamate receptor complex (AMPAR), in single synapses and across dendrites
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